In the mouse visual system, axons of retinal ganglion cells (RGCs) exit from each eye and converge at the ventral midline of the brain to form the optic chiasm (OC), then grow to visual centers in the thalamus, where they synapse onto second-order neurons that project to the cerebral cortex. In animals with frontally located eyes, some RGC axons do not cross the midline and instead project to the ipsilateral, or same-side, visual centers. This circuit mediates binocular vision and we have long focused on the formation of this circuit. The present proposal addresses how ipsilateral (ipsi) and contralateral (contra) RGCs axons extend together in common tracts through the developing brain, converging and diverging throughout their journey to the target, and whether such cohort integrity in paths is relevant to connectivity within target regions. In this proposal, we seek to more fully characterize eye-specific axonal cohort maneuvers in the pathway from eye through the OC to first target. In Aim 1, we will investigate the organization of the ipsi and contra axons, viewed with different tracers in the same preparation, focusing on where the ipsi and contra RGC axons from the same and opposite eyes interleave and where they segregate. We will use classical and novel morphological analysis at the LM and EM levels to chart fasciculation of ipsi and contra RGCs along the path and their relationships with glial cells. We will also devise explant assays to dissect the cellular and molecular mechanisms for fiber-fiber interactions during eye-specific axon pathway formation. In Aim 2, we will investigate the effects of mutations that have known effects on decussation and targeting on RGC eye- specific fiber organization. Throughout, we will compare and contrast the cell and molecular differences of ipsi and contra RGCs as they diverge amongst radial glia at the optic chiasm midline, associate with their own and opposite eye cohorts, exit the chiasm and grow in the tract where astroglia are organized to potentially influence their trajectory, and enter and terminate in the dorsal lateral geniculate nuclei (dLGN).. Approaches developed over the last few decades in our lab will facilitate analysis of axons at the single fiber and cohort levels, aided by a newly developed clearing technique and special microscopy, and electroporation in utero for delivery of GFP-constructs into the retina. We will address the relationship between decussation, eye-specific organization within the path, and early target innervation, linking phases of axon guidance that were previously addressed as discrete steps and restricted to focus on a single cohort. Understanding how tracts are laid down is essential for unraveling the phenotypes of neurological disorders, in which defects in fiber pathways and synapse formation are implicated but their relationship is not fully understood. Moreover, implementing axon regeneration depends on knowledge of how the entire pathway is organized and to what extent each phase of axon organization prepares axons for the next phase.